Development of an Anti-canine PD-L1 Antibody and Caninized PD-L1 Mouse Model as Translational Research Tools for the Study of Immunotherapy in Humans

Immune checkpoint blockade therapy, one of the most promising cancer immunotherapies, has shown remarkable clinical impact in multiple cancer types. Despite the recent success of immune checkpoint blockade therapy, however, the response rates in patients with cancer are limited (∼20%–40%). To improve the success of immune checkpoint blockade therapy, relevant preclinical animal models are essential for the development and testing of multiple combination approaches and strategies. Companion dogs naturally develop several types of cancer that in many respects resemble clinical cancer in human patients. Therefore, the canine studies of immuno-oncology drugs can generate knowledge that informs and prioritizes new immuno-oncology therapy in humans. The challenge has been, however, that immunotherapeutic antibodies targeting canine immune checkpoint molecules such as canine PD-L1 (cPD-L1) have not been commercially available. Here, we developed a new cPD-L1 antibody as an immuno-oncology drug and characterized its functional and biological properties in multiple assays. We also evaluated the therapeutic efficacy of cPD-L1 antibodies in our unique caninized PD-L1 mice. Together, these in vitro and in vivo data, which include an initial safety profile in laboratory dogs, support development of this cPD-L1 antibody as an immune checkpoint inhibitor for studies in dogs with naturally occurring cancer for translational research. Our new therapeutic antibody and caninized PD-L1 mouse model will be essential translational research tools in raising the success rate of immunotherapy in both dogs and humans. Significance: Our cPD-L1 antibody and unique caninized mouse model will be critical research tools to improve the efficacy of immune checkpoint blockade therapy in both dogs and humans. Furthermore, these tools will open new perspectives for immunotherapy applications in cancer as well as other autoimmune diseases that could benefit a diverse and broader patient population.


Introduction
Immune checkpoint blockade therapy, one of the most promising forms of cancer immunotherapy, has been successful in multiple cancer types, including invasive urinary bladder cancer, the focus of this study (1,2). In particular, programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (4,5). Although this worthy milestone conveys the excitement and promise of this novel form of cancer treatment, PD-1/PD-L1 blockade therapy in cancer is currently not satisfactory due to the limited response rates (20%-40%; refs. [3][4][5]. Therefore, new immunotherapeutic strategies to improve the efficacy of current PD-1/PD-L1 blockade therapies are urgently needed. Strategies to improve PD-1/PD-L1 blockade therapies in bladder cancer and other cancers include: (i) identifying host factors including genetics, immune state, and molecular subtype that drive a relevant response, (ii) assessing biomarkers and combinations of biomarkers to predict response and to personalize therapy, (iii) developing better tools to monitor immune effects, and (iv) selecting combination drug approaches/regimens to address multiple "defects" in the immune response in addition to PD-1/PD-L1 blockade. Relevant preclinical animal models are essential to developing these strategies and testing multiple combination approaches. Factors that are likely to affect the PD-1/PD-L1 axis and thus must be represented in animal models, include aggressive and metastatic cancer behavior, tumor heterogeneity, mutational landscape, genetic and epigenetic cross-talk, cancer molecular subtypes, immune cell responsiveness, and innate and acquired mechanisms of drug resistance. Experimental rodent models, including carcinogen-induced, engraftment, and genetically engineered models, are instrumental in research of different types of cancer including bladder cancer (6)(7)(8)(9). However, rodent models lack the collective features that are critical to studying emerging therapies within and across molecular subtypes in bladder cancer, and to predicting therapeutic success or failure in humans.
While these collective features are lacking in rodent models, we have demonstrated that pet dogs with naturally occurring invasive urothelial carcinoma (InvUC; comprising >90% of bladder cancer in dogs) can provide this crucially needed relevant model in an immunocompetent host. The canine model can complement other models to drive preclinical research to understand and optimize drug activity in humans. Canine InvUC mimics human InvUC in presentation, pathology, local invasion, distant metastases (lung and other organs in >50% of cases), and chemotherapy response (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Canine and human InvUC are similar in terms of druggable mutations, pathway variants, epigenetic targets, and transcriptomic patterns of molecular subtypes (basal, luminal; refs. 11, 13, 18, 21-27). InvUC represents 1.5%-2% of the estimated 4 million new cases of canine cancer annually in the United States, so ample numbers of dogs are available for translational studies (28). Canine clinical trials in which dogs continue life as pets are a win-win situation with benefits to each dog and knowledge gained to help people and pet dogs facing cancer (11,14,18). Thus, dogs offer an excellent opportunity to advance PD-1/PD-L1 blockade therapies in humans. Successful treatment approaches in rodents can be evaluated in dogs, and those that have the highest success can be moved into human trials.
Canine PD-1/PD-L1 blockade antibodies are not commercially available for dogs with InvUC. The development of canine PD-L1 (cPD-L1) antibodies has been described by several academic groups (29)(30)(31)(32)(33). Tumor regression in dogs with oral melanoma and soft-tissue sarcomas was reported in response to a canine chimeric mAb targeting PD-L1 (30,33). The antitumor effects of this antibody, however, require further study as the effect of concurrent medications (30,34,35) on the tumor regression was not determined. In other work, Choi and colleagues developed anti-canine PD-1 and PD-L1 antibodies for diagnostic applications, but these have not been used therapeutically (31). Therefore, development of a canine immune checkpoint blockade antibody, that is, an anti-cPD-L1 antibody, for dogs is important for translational research.
In this study, we developed a new immunotherapeutic cPD-L1 antibody and characterized its functional/biological properties in vitro and in vivo. Furthermore, we validated the therapeutic efficacy of cPD-L1 antibodies using our newly established caninized PD-L1 mice, which express cPD-L1 on the cell surface. Our cPD-L1 antibody, a new immuno-oncology drug, and caninized PD-L1 mice will be essential translational research tools in raising the success rate of immunotherapy in dogs and humans.

Study Overview
The work included: (i) generation of cPD-L1 antibodies in mice, (ii) selection of the superior clone to further develop, (iii) evaluation of the cPD-L1 antibody in mice expressing cPD-L1 on the cell surface and implanted with tumor cells expressing canine PD-1, (iv) creation of a chimera antibody of the top clone, (v) characterization of the chimera antibody through cell-based and cell-free cPD-L1 antibody binding and cPD-L1/cPD-1 blockade assays, and other assays of essential characteristics, (vi) assessment of the antibody activity in an ex vivo canine peripheral blood mononuclear cell (PBMC)-mediated killing assay, and (vii) initial safety and pharmacology study in dogs, and is summarized in Fig. 1. myeloma cells (see Fig. 1). Supernatants from isolated clones were screened for the ability to block the cPD-1/cPD-L1 interaction through cPD-L1-expressing cell-based ELISAs (see Fig. 2A and B for details), with the mAbs 3C8D3 (3C) and 12C10E4 (12C) selected for further study. Clonal antibodies were purified from supernatants and the same assays were repeated.

Generation of the Canine CD274 Knock-in Mouse
The caninized PD-L1 mouse (canine CD knock-in mouse) was generated by Easi-CRISPR (Efficient additions with ssDNA insert-CRISPR) strategy using a long single-strand DNA (ssDNA) donor and CRISPR ribonucleoproteins (39). Briefly, the long ssDNA (a full-length of canine CD cDNA; NM_001291972) was injected with preassembled guide RNA (gRNA, CAGCAAATATCCTCATGTTT TGG) and Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. The ssDNA and single-guide RNA were synthesized at Integrated DNA Technologies. C57BL/6N female mice at 4 weeks of age (Envigo) were superovulated and then mouse zygotes were obtained by mating C57BL/6N males with the superovulated females.

Immunofluorescence Study of Mouse Tumor Tissues
Tumor masses were frozen in optimal cutting temperature blocks immediately after excision. Cryostat sections of 5-μm thickness were attached to  Fc (hFc) protein, Alexa Fluor 488-conjugated anti-human IgG Fc-specific secondary antibody and/or cPD-L1 antibody were added, and then green fluorescence signal was measured to quantify the amount of bound PD-1 protein by IncuCyte S3. E, A representative result of the cPD-L1/cPD-1 blockade assay. Kinetic graphs from each well of a 96-well plate showing quantitative binding of cPD-1 protein on BT549 cells expressing cPD-L1 at 3-hour intervals after the addition of cPD-L1 antibodies. The positive clones that blocked the interaction of cPD-L1/cPD-1 proteins are highlighted in red (A4 and B8). F, Representative images (at 18 hours) of the cPD-L1/cPD-1 blockade. Green fluorescent merged images of cPD-L1-expressing cells are shown. Note the lack of fluorescence due to the antibody binding to PD-L1 and blocking the interaction with cPD-1.
saline-coated slides. Cryostat sections were fixed with 4% paraformaldehyde for 30 minutes at room temperature and blocked with blocking solution (1% BSA, 2% donkey and/or chicken serum, and 0.1 mol/L PBS) at room temperature for 30 minutes. Samples were stained with primary antibodies against CD8 and granzyme B overnight at 4°C, followed by secondary antibodies at room temperature for 1 hour. Nuclear staining was performed with Hoechst 33342 (Thermo Fisher Scientific). The

Expression and Purification of a Recombinant cPD-L1 Antibody, 12C10E4
The codon optimized for CHO variable light (VL) and heavy (VH) chains were cloned into pTRIOZ-hIgG1 vector (InvivoGen), and then the constant light and heavy chains were replaced with canine kappa light constant chain and canine IgG2 heavy constant chain: pTRIOZ-cIgG2-12C10E4. Plasmids encoding 12C10E4 chimeric antibody, pTRIOZ cIgG2 12C10E4, were transfected into ExpiCHO-S cells following the transfection kit instructions (GIBCO, A29133). ExpiCHO-S cells were cultured with ExpiCHO Expression Medium (Thermo Fisher Scientific) in a shaker incubator set at 120 rpm, 37°C and 8.0% CO 2 . Cells were collected 10 days posttransfection by centrifugation at
Every hour, green fluorescent signal was measured and quantified by IncuCyte S3 (Sartorius). To measure PD-1 protein on the cells, we seeded 1 × 10 4 BT549 cPD-L1 cells per well in 96-well plates, and then incubated the plates with cIgG control (Rockland Immunochemicals), or 12C10E4 antibody, cPD-1-hFc protein (human Fc protein conjugated; SinoBiological US), and/or anti-human Alexa Fluor 488 dye conjugate (Thermo Fisher Scientific). Every 3 hours, green fluorescent signal was measured and quantified by IncuCyte S3 (Sartorius). The Image analysis was performed according to the manufacturer's protocol.

Flow Cytometry Analysis
MB49, MB49 cPD-L1 , K9TCC, or K9TCC nRFP cells were washed twice with icecold cell staining buffer (BioLegend) and stained with cIgG control or 12C10E4 cIgG for 1 hour at 4°C. After three washes with staining buffer, cell samples were stained with Alexa Fluor 488-conjugated anti-canine IgG-specific secondary antibody for 30 minutes at 4°C. Cell samples were loaded on BD LSRFortessa (BD) for analysis. Data analysis was performed on FlowJo v9 software (BD).

Binding Affinity (K D ) Determination
The binding affinity (K D ) of cPD-L1 protein and cPD-L1 antibody (12C10E4) was determined by Octet Biolayer interferometry using the Octet RED384 system (Sartorius). Briefly, His-tagged cPD-L1 protein was loaded on the Octet NTA biosensor at a concentration of 200 nmol/L. The association step was performed by submerging the sensors in three concentrations of the 12C10E4 antibody (50, 100, 200 nmol/L) in the kinetic buffer. Dissociation was performed and monitored in fresh kinetic buffer. Data were analyzed with Octet Analysis HT software (Sartorius).

SDS-PAGE and Isoelectric Focusing
The purity and isoelectric point (pI) of the purified antibodies were determined by SDS-PAGE and isoelectric focusing (IEF), respectively. SDS-PAGE or IEF gels were purchased from Bio-Rad Laboratories or Thermo Fisher Scientific. The SDS-PAGE, IEF, and Coomassie blue staining were performed according to the manufacturer's protocol. Image acquisition and quantitation of band intensity were performed using Odyssey CLx infrared imaging system (LI-COR Biosciences).

Size Exclusion Chromatography
Size exclusion chromatography (SEC) analysis was performed to detect antibody aggregates and monomers. The AKTA Pure 150 M (Cytiva) and Superdex 200 Increase 10/300 GL column (Cytiva) were used to analyze antibodies at a flow rate 0.3 mL/minute for 135 minutes. Elution was monitored using UV absorption at 280 nm, and data were processed by Unicorn 7 software (Cytiva). The SEC analysis was performed in the Molecular Evolution, Protein Engineering, and Production core facility at Purdue University (West Lafayette, IN).

Peptide Mapping Analysis
The peptide mapping comparison for each 12C10E4 batch was performed.
Briefly, the antibody was enzymatically digested with trypsin on S-trap micro columns from Protifi after reduction and alkylation (42). Peptides were then separated and analyzed by a reversed-phase liquid chromatography tandem mass spectrometry (RP-LC/MS-MS) using a Q Exactive HF Hybrid Quadrupole-Orbitrap MS (Thermo Fisher Scientific) equipped with a Nanospray Flex Ion Source (Thermo Fisher Scientific), coupled with a Dionex UltiMate 3000 RSLC Nano System (Thermo Fisher Scientific). The resultant mass spectrometric data were analyzed using the PEAK PTM workflow in the PEAKS X PRO Studio 10.6 software package from Bioinformatics solutions Inc.
to map the detected MS1 and MS2 ions to the amino acid sequence of antibody (43). The peptide mapping analysis was performed in the Bindley Bioscience Center Purdue Proteomics Facility at Purdue University (West Lafayette, IN).
LC/MS-MS data were used for mapping glycosylation (0.98 Da) of asparagine (N) and glutamine (Q) residues of the mapped antibody sequences.

N-Glycomic Analysis of 12C10E4 Antibody
N-glycomic analysis of 12C10E4 antibody was performed by methods described previously (44). Briefly, N-glycans of 12C10E4 antibody were released by treating the reduced and alkylated protein with PNGase F. The released N-glycan fractions were then permethylated. The permethylated N-glycans were evaluated by Matrix-assisted laser desorption/ ionization -mass spectrometry (MALDI-MS) using the AB SCIEX TOF/TOF 5800 mass spectrometer (Applied Biosystem/MDS Analytical Technologies). The structural assignments of the N-glycans were based on molecular weight and followed the principles of the N-glycan biosynthesis pathway. The carbohydrate analysis was performed at the Complex Carbohydrate Research Center, the University of Georgia (Athens, GA; supported by NIH R24GM137782 grant).
MILLIPLEX Canine Cytokine/Chemokine Magnetic Bead Panel (Millipore) was used to multiplex and measure IFNγ, IL10, and TNFα in these activated canine PBMCs following manufacturer's protocols. Samples were incubated with the cytokine magnetic beads on shaker for 2 hours followed by incubation with secondary detection antibody provided in the kit. The plate was read on an Attune flow cytometer (Thermo Fisher Scientific) by using the FL2 (PE channel) channel for the reporter and FL4 (APC channel) for classification. For each of the cytokines, 300 beads were measured, and data were collected as a forward and side scatter dot plot. Concentrations of cytokines were quantified as ng/mL using Cytokine Multiplex Analysis Software (MPLEX, Cytomics Analytical LLC). The data show a significant induction in the expression of IFNγ, IL10, and TNFα with anti-CD3/CD28 and IL2 treatment as compared with IL2 treatment alone, and this activation protocol was used for the remainder of the work.

NanoString Analysis of Activated cPBMCs
RNA was isolated from activated cPBMCs as described previously (RNeasy kit, Qiagen) and submitted to the Stark Neurosciences Research Institute Biomarker Core, Indiana University School of Medicine, Indiana University, Indianapolis, IN, for detection of modulation of genes upon activation using the nCounter Canine IO Panel (NanoString Technologies). Data were analyzed using Rosalind (Rosalind). Groupwise comparison was conducted using control cPBMCs and compared with activated cells from three dogs. Differentially expressed genes (Fold change (FC) ≥ 1.5; P < 0.05) were considered significant. Data were visualized using heatmap, volcano plot, and histogram for specific genes.

Tumor Cell Killing Assay
The tumor cell killing assay was performed according to the previous description (48

Pilot Study in Laboratory Dogs
A single-dose pilot study to assess initial safety and pharmacokinetic parameters was performed in six laboratory beagles approximately 12

Detection of the cPD-L1 Antibody in Dog Serum
The

Statistical Analysis
All quantitative results were displayed as the mean ± SD, with at least three biological replicates. The intergroup statistical significance was calculated by two-tail Student t test. P < 0.05 was considered statistically significant.

Data Availability Statement
The data generated in this study are available upon request from the corresponding author.

Development and High-throughput Screening of Anti-cPD-L1 Antibodies
Anti-cPD-L1 mAbs were successfully generated using conventional hybridoma procedures (50). Please see Fig. 1, which summarizes an overview of cPD-L1 antibody development. Antibodies were screened using PD-L1 binding and blockade assays similar to those we described previously (refs. 37, 41, 48; Fig. 2A and B). Among over 2,000 hybridomas, 154 clones were selected against membranelocalized cPD-L1 protein in a live cell-based antibody binding assay ( Fig. 2C and D). Of these, 10 clones were found to block the cPD-L1/cPD-1 interactions ( Fig. 2E and F we selected the antibodies termed 3C8D3 (3C) and 12C10E4 (12C) for further analysis.

Evaluation of Therapeutic Efficacy of cPD-L1 Antibodies in the Caninized PD-L1 Mice
To further assess the clinical use of the cPD-L1 antibody as an immunotherapeutic drug, its therapeutic efficacy needed to be evaluated in an appropriate in vivo model. To do so, we established caninized PD-L1 (C57BL/6 background) mice. Briefly, we generated mice that expressed cPD-L1 on the cell surface by replacing the mouse Cd with canine CD using a CRISPR knock-in mouse strategy (Fig. 3A). To evaluate the therapeutic efficacy of the cPD-L1 antibodies in a syngeneic animal model, we generated the mouse bladder cancer cell line MB49-expressing cPD-L1 (MB49 cPD-L1 ) by knocking out mPD-L1 and reexpressing cPD-L1 ( Fig. 3B and C). Although MB49 cPD-L1 and the caninized PD-L1 mice express cPD-L1 protein instead of mPD-L1 protein, these caninized PD-L1 mice express mPD-1 protein. Therefore, we examined whether cPD-L1 protein interacts with mPD-1 protein before evaluating the therapeutic efficacy of the cPD-L1 antibody in the caninized PD-L1 mice. The binding of cPD-L1 and mPD-1 was similar to the cognate cPD-L1 and cPD-1 pair (Fig. 3D). Consistently, the cPD-L1 antibody (12C) efficiently blocked both the cPD-L1/mPD-1 and cPD-L1/cPD-1 interactions, but not that of mPD-L1/mPD-1 or mPD-L1/cPD-1 as the cPD-L1 antibodies do not recognize mPD-L1 ( Fig. 3D and E).
Treatment of MB49 cPD-L1 tumors in the cPD-L1 mice with either the 12C or 3C antibody significantly reduced the tumor size (Fig. 3F), and increased the number of infiltrating cytotoxic T cells relative to mice treated with control IgG as measured by CD8 and granzyme B expression (Fig. 3G-I). Both the 12C and 3C antibodies demonstrated good safety profiles in mice in that body weight was maintained, and there were no changes in kidney function as assessed by serum creatinine or liver enzyme activity ( Fig. 3J and K). The in vitro and in vivo validation results indicated that the cPD-L1 antibodies that recognize cPD-L1 effectively inhibit the PD-1/PD-L1 pathway and enhance mouse antitumor immunity.

Characterization and Evaluation of the Caninized cPD-L1 Chimeric Antibody as a New Immunotherapeutic Antibody
For clinical use of cPD-L1 antibodies in dogs, the 12C antibody was caninized by replacing the mouse constant domain with canine IgG2 (equivalent to human IgG1) constant domains. Briefly, we sequenced full-length VH and VL RNA transcripts obtained from hybridoma clones by 5 /3 RACE and cloned these into the pTRIOZ-cIgG2-ck vector, which is designed for high-yield production of whole mAbs from a single plasmid (Fig. 4A). The chimeric cPD-L1 antibody retained the cPD-L1 binding VH and VL chains of the mouse hybridoma.
To monitor batches during antibody production, we identified the attributes of the purified chimeric antibodies, such as purity, pI value, amino acid sequence, and N-glycomic profile (Fig. 4B-F). The chimeric antibody, 12C10E4-cIgG, bound to the membrane-localized cPD-L1 protein ( Fig. 5A and B), but did not recognize cPD-L2 protein (Fig. 5C). The affinity (K D ) of the chimeric antibody as determined by Octet was 8.6 nmol/L (Fig. 5D). Similar to the murine 12C antibody obtained from the hybridoma, the 12C chimeric antibody blocked the cPD-L1/cPD-1 interaction (EC 50 = 0.419 μg/mL; Fig. 5E).
To demonstrate immune checkpoint inhibition of the 12C chimeric antibody, an ex vivo canine system such as a tumor cell killing assay in which cPD-L1-positive canine bladder cancer cells (K9TCC) are cocultured with activated canine PBMCs was established. The canine immune-oncology panel analysis ( Fig. 5F and G; Supplementary Table S1) and analysis of secreted cytokines (IFNγ and TNFα; Fig. 5H and I) demonstrated the activation of canine PBMCs by anti-canine CD3 and CD28 antibodies and canine IL2 treatment. To quantify the number of surviving or dead tumor cells in a tumor cell killing assay, we established K9TCC nRFP cells expressing nuclear-restricted RFP (nRFP) and confirmed the expression of endogenous cPD-L1 protein and mRNA in both K9TCC parental and K9TCC nRFP cells upon IFNγ treatment (Fig. 5J-L). We used these activated canine PBMCs and K9TCC nRFP cells to perform a tumor cell killing assay. Although the PBMC and tumor cells were from different dogs, and thus the dog lymphocyte antigen (DLA) was not matched between the cPBMCs and K9TCC cells, the 12C chimeric antibody enhanced tumor cell killing activity and IFNγ secretion ( Fig. 5M and N).
To study the half-life of the 12C chimeric antibody in dogs, we established a new ELISA-type assay using purified his-tagged cPD-L1 protein (cPD-L1-His) and measured the concentration of 12C chimeric antibody in dog serum (Fig. 6A). In an initial single-dose pharmacology study in beagle dogs, the 12C chimeric antibody was well tolerated and had a half-life of 1 to 2 days ( Fig. 6B and C; Table 1). Interestingly, the half-life of the cPD-L1 antibody was shorter than that for human checkpoint inhibitors and indicated that weekly dosing could be appropriate in dogs. The possible infusion reaction in one dog resolved without intervention. There was good antibody tolerability of the antibody in this single-dose study in the lab dogs and body weight was maintained (Table 1). Nonspecific changes such as a slight reduction in monocyte count reduction and a slight increase in CO 2 and gamma-glutamyl transferase (GGT) were transient and resolved without intervention (Table 1).

Discussion
Companion dogs naturally develop several types of cancer that in many respects resemble clinical cancer in human patients (51). Although mouse models are the most commonly used animal model in cancer research, they do not possess collective features such as tumor heterogeneity, mutational landscape, cancer molecular subtypes, and immune cell responsiveness present in human cancer (7). Therefore, studies in mouse models should be complemented by other models such as specific forms of naturally occurring cancer in pet dogs (51). These complementary studies are especially important for the development or study of new immuno-oncology drugs like novel immune checkpoint inhibitors (ICI) or of combination regimens for companion dogs that can develop naturally occurring cancer in the context of an intact immune system and an aggressive heterogeneous cancer. The development of canine ICIs is expected to expand comparative oncology approaches to improve the current therapeutic efficacy of immunotherapies in human cancer. Therefore, the canine studies of immuno-oncology drugs produce translatable knowledge that can inform and prioritize new immuno-oncology therapy in humans. The challenge has been, however, that ICIs that target canine immune checkpoint molecules such as cPD-1 and cPD-L1 have not been commercially available.
We successfully developed a new cPD-L1 antibody as an immuno-oncology drug and characterized its functional and biological properties using multiple assays including a cPBMC-mediated canine tumor cell killing assay. It is recognized that in this assay ( Fig. 5M and N), the increase in tumor cell killing activity associated with the 12C antibody may not directly represent T cellmediated activity due to an unmatched DLA between cPBMCs and K9TCC the initial safety profile in the laboratory dogs (Table 1), our cPD-L1 antibody is a promising ICI for dogs. We anticipate that our chimeric cPD-L1 antibody will be an efficacious immune checkpoint blockade antibody for patients with canine cancer.  Slightly high CO 2 (25 mmol/L, reference range 13-24 mmol/L) at 1 week after treatment; normalized by 2 weeks after treatment in 1 dog and by 4 weeks after antibody treatment in a second dog (1 dog, 2 mg/kg and the second dog 5 mg/kg cPDL-1 antibody).

Other observations
Possible infusion reaction in 1 dog who experienced weakness, pale mucous membranes, and bradycardia (reduced heart rate) that started 5 minutes after the completion of the antibody infusion. The dog returned to normal within 10 minutes with no intervention (1 dog, 5 mg/kg cPD-L1 antibody). Mildly decreased appetite the day of treatment in 3 dogs (2 dogs, 2 mg/kg cPD-L1 antibody; 1 dog 5 mg/kg cPD-L1 antibody).
Other than these findings, the dogs remained bright, alert, and active, maintained body weight, and had normal temperature/pulse/respiration.
Because treatment of canine cancers with anti-PD-L1 or anti-PD-1 antibodies is a fairly unexplored area, there were some limitations in these initial studies such as sample size as well as variation in study cases such as cancer type and stage, and status of prior treatment (i.e., treatment-naïve vs. history of prior treatment), which could have contributed to the inconsistency and variation in outcomes. Nonetheless, both groups demonstrated the potential of these antibodies to treat certain cancer types, especially OMM, with positive responses noted (30,32). In addition, the mAbs designed by Choi and colleagues further supported the ability of anti-PD-L1 antibodies to augment IFNγ secretion in PBMC cultures that is suggestive of the potential of PD-L1 blockade to reinvigorate T-cell activity in canine tumors (31). Recently, Maekawa and colleagues further studied their c4G12 anti-PD-L1 antibody in a group of dogs with primary OMM and, consistent with responses in human studies, observed good safety with no serious adverse events recorded (33). They did observe antitumor responses in some dogs in the treatment group, and the study outcomes were suggestive of enhanced survival with treatment compared with the control group (33). Despite limitations associated with the nature of the immunotherapy administration for treatment of canine cancers and the relatively recent and novel nature of these treatments, the studies discussed support the clinical promise of antibodies targeting the PD-1/PD-L1 axis and the need for further studies. Considered together with antibodies described by other groups, our new cPD-L1 antibody will broaden treatment options for patients with canine cancer and could provide clinical benefits similar to those offered by the human PD-L1 antibodies, atezolizumab, durvalumab, and avelumab that have undergone extensive clinical assessment.
In the development of immuno-oncology drugs, particularly immunotherapeutic antibodies, translation of discoveries in mouse models to clinical trials has been hindered by multiple biological differences between mice and humans, such as the lack of cross-reactivity between species. For example, if an antihuman or cPD-L1 antibody does not recognize mPD-L1 protein, the therapeutic efficacy of that antibody cannot be evaluated in a syngeneic mouse model. Mice with an engrafted human immune system have been developed for translational research to help overcome this constraint. Indeed, pembrolizumab, an anti-human PD-1 antibody, showed tumor growth inhibition and CD8 + T-cell activation in humanized NSG mice that received tumor implants from patientderived xenografts (59). Despite the importance of tumor-bearing mice with engraftment of a human immune system for preclinical immuno-oncology research, this model nonetheless presents considerable obstacles such as a limited source of human cells and tissues, immune rejection, and high cost (60). As an alternative mouse model for immunotherapeutic antibody development, humanized immune checkpoint mice are commercially available. For example, humanized PD-L1 mice have been generated by replacing the mPD-L1 gene (Cd) with the human PD-L1 gene (CD) using CRISPR/CAS9 methods.
The humanized PD-L1 mouse can be used to evaluate the therapeutic efficacy of anti-human PD-L1 antibodies in vivo. However, no caninized PD-L1 mouse model has been previously reported. The lack of a suitable mouse model is a major obstacle for developing canine ICIs such as PD-1/PD-L1 blockade antibodies, in which the ideal approach would be to move successful treatment approaches in mice to studies in dogs, and to further translate those with the highest success in dogs into human trials. To overcome this obstacle, we established a caninized PD-L1 mouse model as a preclinical tool, and validated the therapeutic efficacy of immunotherapeutic cPD-L1 antibodies in vivo (Fig. 3). Our caninized PD-L1 mouse model and syngeneic mouse bladder cancer cell line, MB49 cPD-L1 , are unique and powerful tools for preclinical canine immuno-oncology research.
In conclusion, our cPD-L1 antibody and unique caninized mouse model will be critical research tools to improve the efficacy of immune checkpoint blockade therapy in both dogs and humans. Furthermore, these tools will open new perspectives for immunotherapy applications in cancer as well as other autoimmune diseases that could benefit a diverse and broader patient population.